Mechansims of hormone receptor signal transduction by G proteins

The overall goal of the Garrison research program is to undestand how the large number of G protein a and bg isoforms lead to specificity in cell signaling, especially how the multiple isoforms of the bg dimer selectively regulate signaling. The bg dimer plays a major role in activating cerain  hematopoietic cells and they are attempting to understand the interaction between two major signaling pathways in the cell membrane, one utilized by Gi linked receptors to activate hematopoietic cells and another used by Gs linked receptors to inhibit many of the inflammatory responses of these cells. In neutrophils and macrophages, activation of Gi coupled receptors such as the f-Met-Leu-Phe or LPA receptors and release the G protein bg dimer. This response activates superoxide production, cell shape changes and cell migration. Cellular targets for Gbg dimers include phosphatidylinositol 3-kinase (PI 3-kinase) and the Rac guanine nucleotide exchange factor (GEF), P-Rex1. Activation of PI 3-kinase generates PIP₃ in the plasma membrane and Rac is a central regulator of cell shape changes and cell migration. As P-Rex1 activation is modulated by PIP₃ and the G protein bg subunit, the synergistic actions of the Gbg dimer on PI 3-kinase and P-Rex1 combine to activate cells such as neutrophils and macrophages. 

G protein coupled receptors such as the b-adrenergic and adenosine A2a receptors raise cyclic AMP levels in hematopoietic cells and markedly inhibit their activation. As a major target of cyclic AMP in cells is the cyclic AMP depended protein kinase (PKA),  phosphorylation of important regulatory sites in hematopoietic cells by PKA must be central to the inhibitory response. The lab has discovered that both PI 3-kinase and P-Rex1 can be phosphorylated in vitro by PKA with a marked reduction in their activity. Their work examines the ability of pure G protein a?and bg subunits to regulate PI 3-kinase and P-Rex1 in synthetic lipid vesicles containing PIP₃ and PI 3-kinase or Rac to determine which G protein subunits regulate the activity of these enzymes. They are also examining the phosphorylation of pure, recombinant PI 3-kinase and P-Rex1 with the cyclic AMP depended protein kinase and measuring the effect of phoshorylation on their activity in the presence of PIP₃  and the G protein a and bg? subunits. Finally, they are examining how PI 3-kinase and P-Rex1 respond to activation of Gi and Gs coupled receptors in HEK-293 cells, macrophages and neutrophils. They are also using small, inhibitory RNA's delivered to these cells by transfection or stable infection with lentiviruses to determine which isoforms of G proteins regulate PI 3-kinase and P-Rex1 activity in a cellular context. As bg? dimers regulate many other effectors in cells including adenylyl cyclase, PLC-b ion channels, they are examining the effect of suppressing individual G protein a and bg?subunits on these effectors as well.